The Australian Wine Research Institute Varietal Thiols and Green Characters Natoiya Lloyd [email protected] Compounds responsible for the The Australian Wine green character Research Institute Methoxypyrazines N OMe . IBMP, SBMP, IPMP N Sulfur compounds 3-Isobutyl-2-methoxypyrazine . DMS, DES, DMDS (IBMP) . 2-Isobutylthiazole S C6 compounds Dimethyl sulfide . (Z)-3-Hexen-1-ol (DMS) . (E)-2-Hexenal . (Z)-3-Hexenal OH . Hexanal . 1-Hexanol (Z)-3-Hexen-1-ol . Hexyl esters (cis-3-Hexen-1-ol) Grape sources of C6 flavours in wine The Australian Wine Research Institute cis-3-Hexen-1-ol precursors . Formed from unsaturated fatty acids after berry damage (usually upon crushing) . Derived from linolenic acid through enzyme cascade O HO LOX/HPL O ADH OH Linolenic Acid (Z)-3-Hexenal (Z)-3-Hexen-1-ol Lipoxygenase → hydroperoxide lyase → alcohol dehydrogenase The Australian Wine Modulating factors – cis-3-Hexen-1-ol Research Institute Viticulture OH (Z)-3-Hexen-1-ol . Enzymatic formation via LOX pathway leads to C6 compounds (cis-3-Hexen-1-ol) . Differs between varieties and during berry development (e.g. Riesling vs Cabernet Sauvignon) . Highest at pre-veraison in line with unsaturated fatty acid levels – decline in linolenic acid with ripening . Higher in skin (from press cake) than must at all ripening stages The Australian Wine Modulating factors – cis-3-Hexen-1-ol Research Institute OH Winemaking (Z)-3-Hexen-1-ol (cis-3-Hexen-1-ol) . Time and temperature of skin contact – similar extraction from 15-28 oC with max after 10-15 h, continual increase during contact time at 10 oC after 25 h . Relatively stable but SO2 and enzymatic activity have effects – O2 needed for formation . Esterification to the acetate – from green (alcohol) to green/floral/fruity (ester) Storage . Not affected by storage in presence of oxygen . Minimal change with storage on lees for up to seven months . Unaffected by short-term oxidative storage in presence of phenolics . Slow decline with storage for 210 days but no impact from different SO2 levels The Australian Wine Summary Research Institute Green flavours in wine are caused by a number of different compound classes, with vastly different potencies Compound origins are in the grape, often in precursor form Viticultural practices and harvesting decisions can impact on green flavours Green flavours may be desirable, adding complexity or typicity to wine styles # 4 The Australian Wine Sensory impact of cis-3-Hexen-1-ol Research Institute OH (Z)-3-Hexen-1-ol cis-3-Hexen-1-ol – cut grass, herbaceous, leafy; 400 μg/L (cis-3-Hexen-1-ol) threshold Typically not found above threshold in most studies Found in wine up to . 650 µg/L in young red wines (highest in Tempranillo) . 800 µg/L in aged red wines . 75 µg/L in Gewurztraminer # 4 . 600 µg/L in some Italian and Spanish white wine varieties (Falanghina and Macabeo) The Australian Wine Varietal thiols – impact odorants Research Institute Polyfunctional thiols are especially potent and have some of the lowest aroma thresholds of any food odorant Varietal thiols are important impact odorants in some wines e.g. Sauvignon Blanc Thiol Perception Aroma OAV threshold 4-MMP 3 ng/L blackcurrant Up to 30 box tree passionfruit 3-MH 60 ng/L grapefruit Up to 210 passionfruit passion-fruit 3-MHA 4 ng/L Up to 195 box tree sweaty Darriet et al. Flavour Fragr. 1995, 10, 385-392 Tominaga et al. Vitis 1996, 35, 207-210 8 Tominaga et al. Flavour Fragr. 1998, 13, 159-162 The Australian Wine Volatile thiol sensory descriptors Research Institute Individual volatile thiols contribute tropical aromas to wine, 3MH also citrus aroma Volatile thiol combinations had aromas of tropical & cooked green vegetal at both levels, and at high levels also cat urine/sweaty 4MMP does not contribute any distinctive sensory properties at high levels At high concentrations 3MHA is responsible for cat urine/sweaty aromas The Australian Wine Consumer preference of volatile thiols Research Institute There was an optimal level of cat urine/sweaty attribute for one group of consumers identified The majority of consumers preferred the samples with ‘green’ attributes, with a minority strongly preferring the ‘fruit’ and ‘estery’ flavours Clear linking of volatile thiols in Sauvignon Blanc wines, their associated sensory attributes and effects on consumer preference Grape varieties containing volatile thiol The Australian Wine compounds Research Institute White varieties Red varieties Sauvignon Blanc Petit Manseng Cabernet Franc Chardonnay Pinot Blanc Cabernet Sauvignon Chenin Blanc Pinot Gris Grenache Colombard Riesling Merlot Gewürztraminer Scheurebe Pinot Noir Gros Manseng Semillon Koshu Sylvaner Maccabeo Tokay Muscat Muscadet Petit Arvine Volatile thiol concentrations in wines The Australian Wine from around the world Research Institute 500 14000 450 4MMP 3MH 12000 400 350 10000 300 8000 250 6000 200 150 4000 100 concentration3MH (ng/L) 4MMP concentration4MMP (ng/L) 2000 50 0 0 South Africa Australia France New Zealand South Africa Australia France New Zealand (n=12) (n=154) (n=141) (n=136) (n=12) (n=154) (n=121) (n=61) Country of origin Country of origin 4000 3500 3MHA 3000 2500 2000 1500 1000 3MHA concenrtation3MHA (ng/L) 500 0 South Africa Australia France New Zealand (n=12) (n=154) (n=40) (n=136) Country of origin The Australian Wine Varietal thiol formation Research Institute Optimise formation and maximise stability of varietal thiols Need to further understand precursor formation (Stress response : Kobayashi et al) Yeast plays a key role in thiol release into wine Need to understand relationship between precursors and free thiols NH 2 H O HO N OH N O O H O S OH Glut-3-MH O H 2N O H yeast SH yeast S H O S O H O O H CSL 3 -M H ATF 3 -M H A Other Cys-3-MH Intermediates 13 from grapes winemaking The Australian Wine Modulation of volatile thiol precursors Research Institute 3MH precursor Methoxypyrazines 4MMP precursor • 3MH precursors are mainly found in the skins of grape berries • 4MMP precursors are mainly found in the flesh of grape berries The Australian Wine HPLC-MS/MS analysis of precursors Research Institute Amount of precursors measured in SAB juice: Cys-3-MH 21 – 55 µg/L Glut-3-MH 245 – 696 µg/L Also found precursors in other varieties (in the juice) generally: Sauvignon Blanc > Pinot Gris > Chardonnay > Riesling Capone et al. JAFC 2010, 58, 1390-1395 15 Precursor Grape studies The Australian Wine Research Institute 5 different SAB clones in the same location in Adelaide Hills of South Australia . Ripening . Transportation / Holding 16 Concentration of 3MH during ripening: The Australian Wine Clone effects Research Institute 450 400 Q9720 /L) H5V10 ng ( 350 F7V7 3MH 300 5385 250 FV46 200 150 Concentration Concentration of 100 50 0 Veraison Mid Ripening Pre-harvest Harvest After Ferm 17 Capone et al. 2011, JAFC. 59: 4649-4658 Amount of 3-MH precursors during ripening The Australian Wine Research Institute 450 400 (R)-Glut 3-MH 350 (S)-Glut 3-MH 300 (R)-Cys 3-MH 250 (S)-Cys 3-MH 200 150 Concentration (µg/L) Concentration 100 50 0 Clone 1 Clone Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 2 Clone 3 Clone 4 Clone 5 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone Veraison Mid Ripening Pre-harvest Harvest 18 Capone et al. 2011, JAFC. 59: 4649-4658 Effect of transportation on precursor The Australian Wine concentration Research Institute No SO2 No SO2 50 SO2 50 SO2 50 SO2 500 SO2 500 SO2 No Asc 100 Asc No Asc 100 Asc 500 Asc No Asc 500 Asc Analysed shortly after machine harvesting then …….. 19 The Australian Wine Research Institute 794 km 794 km 20 The Australian Wine Research Institute 794 km 794 km 21 The Australian Wine Effect of transportation on precursors Research Institute 1400 Cys-3-MH 1200 Glut-3-MH 1000 800 600 400 200 0 Precursor Concentration Precursor Concentration (nmol/L) 50SO2_noasc 50SO2_noasc noSO2_noasc noSO2_noasc 50SO2_100asc 50SO2_500asc 50SO2_100asc 50SO2_500asc noSO2_100asc 500SO2_noasc noSO2_100asc 500SO2_noasc 500SO2_500asc 500SO2_500asc Prior to Transportation After Transportation 22 Capone et al. 2011, JAFC. 59: 4659-4667 The Australian Wine Modulation of volatile thiol precursors Research Institute • Glutathione 3MH precursor is more abundant than Cysteine 3MH precursor, regardless of grape variety • 3MH precursors are affected by ripening. • 4MMP precursor peaked early in ripening season, at approx. 10°Beame • Mild water stress & moderate Nitrogen supply increased volatile thiols in wine • Foliage Copper spray pre-veraison decreased volatile thiols in wine • Foliar Nitrogen fertiliser with & without Sulfur increased volatile thiols in wine • Botrytis infection affects the levels of volatile thiols in the wine • 4MMP precursor found in free run juice & light pressings • 3MH precursor extracted mainly during skin contact – particularly longer periods of maceration and higher temperatures (18-20°C) Formation pathway to 3-MH The Australian Wine Research Institute 24 Conclusions – Factors affecting precursor The Australian Wine concentration in fruit Research Institute Ripening - Low levels of precursors until commercial harvest Transportation – inc. precursor for Cys and Glut SO2 and Ascorbic acid – a combination of both optimum – very high SO2 suppresses conjugate formation Glut-3-MHAl – tentatively identified as intermediate between (hexenal + glutathione) and Glut-3-MH for the first time Cysgly-3-MH – Confirmed presence, is short lived 25 The Australian Wine Modulation of volatile thiols Research